Skip to main content
PMC home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1984 Nov;81(22):6993–6997. doi: 10.1073/pnas.81.22.6993

Covalent bond formation between a DNA-cytosine methyltransferase and DNA containing 5-azacytosine.

D V Santi, A Norment, C E Garrett
PMCID: PMC392062  PMID: 6209710

Abstract

DNA containing 5-azacytosine (azaC) has previously been shown to be a potent inhibitor of DNA-cytosine methyltransferases. In this report, we describe experiments which demonstrate that azaC-DNA forms a covalent complex with Hpa II methylase, a bacterial enzyme that methylates the internal C of C-C-G-G sequences. The complex does not undergo detectable dissociation over at least 3 days and is stable to denaturation with NaDodSO4. After extensive digestion of the complex with DNase and phosphodiesterase, gel filtration gave the methylase bound to approximately one equivalent of azaC; the digested complex had an apparent molecular weight similar to that of the native enzyme. Although prior treatment of azaC-DNA with Hpa II endonuclease had only a slight effect on binding of the methylase, treatment with Msp I endonuclease, which also cleaves at C-C-G-G sequences, resulted in a significant reduction in binding; this indicates that azaC residues in the recognition sequence of Hpa II are an important component in the covalent interaction of the methylase. However, since there was residual binding it is possible that azaC residues elsewhere in DNA also covalently bind to the methylase. These results provide an explanation of why azaC-DNA is such a potent inhibitor of cytosine methyltransferases and how the incorporation of such low levels of azaC into DNA can result in dramatic decreases in the methylation of cytosine. Finally, consideration of the probable catalytic mechanism of cytosine methylases and the chemical properties of azaC suggests that the inhibition is, at least in part, an active-site directed process and permits a proposal for the structure of the covalent complex.

Full text

PDF
6993

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bodnar J. W., Zempsky W., Warder D., Bergson C., Ward D. C. Effect of nucleotide analogs on the cleavage of DNA by the restriction enzymes AluI, DdeI, HinfI, RsaI, and TaqI. J Biol Chem. 1983 Dec 25;258(24):15206–15213. [PubMed] [Google Scholar]
  2. Compere S. J., Palmiter R. D. DNA methylation controls the inducibility of the mouse metallothionein-I gene lymphoid cells. Cell. 1981 Jul;25(1):233–240. doi: 10.1016/0092-8674(81)90248-8. [DOI] [PubMed] [Google Scholar]
  3. Constantinides P. G., Taylor S. M., Jones P. A. Phenotypic conversion of cultured mouse embryo cells by aza pyrimidine nucleosides. Dev Biol. 1978 Sep;66(1):57–71. doi: 10.1016/0012-1606(78)90273-7. [DOI] [PubMed] [Google Scholar]
  4. Creusot F., Acs G., Christman J. K. Inhibition of DNA methyltransferase and induction of Friend erythroleukemia cell differentiation by 5-azacytidine and 5-aza-2'-deoxycytidine. J Biol Chem. 1982 Feb 25;257(4):2041–2048. [PubMed] [Google Scholar]
  5. Doerfler W. DNA methylation and gene activity. Annu Rev Biochem. 1983;52:93–124. doi: 10.1146/annurev.bi.52.070183.000521. [DOI] [PubMed] [Google Scholar]
  6. Friedman S. The inhibition of DNA(cytosine-5)methylases by 5-azacytidine. The effect of azacytosine-containing DNA. Mol Pharmacol. 1981 Mar;19(2):314–320. [PubMed] [Google Scholar]
  7. Harris M. Induction of thymidine kinase in enzyme-deficient Chinese hamster cells. Cell. 1982 Jun;29(2):483–492. doi: 10.1016/0092-8674(82)90165-9. [DOI] [PubMed] [Google Scholar]
  8. Harrison J. J., Anisowicz A., Gadi I. K., Raffeld M., Sager R. Azacytidine-induced tumorigenesis of CHEF/18 cells: correlated DNA methylation and chromosome changes. Proc Natl Acad Sci U S A. 1983 Nov;80(21):6606–6610. doi: 10.1073/pnas.80.21.6606. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Jones P. A., Taylor S. M. Cellular differentiation, cytidine analogs and DNA methylation. Cell. 1980 May;20(1):85–93. doi: 10.1016/0092-8674(80)90237-8. [DOI] [PubMed] [Google Scholar]
  10. Kreuzer K. N., Alberts B. M. Site-specific recognition of bacteriophage T4 DNA by T4 type II DNA topoisomerase and Escherichia coli DNA gyrase. J Biol Chem. 1984 Apr 25;259(8):5339–5346. [PubMed] [Google Scholar]
  11. Pogolotti A. L., Jr, Santi D. V. High-pressure liquid chromatography--ultraviolet analysis of intracellular nucleotides. Anal Biochem. 1982 Nov 1;126(2):335–345. doi: 10.1016/0003-2697(82)90524-3. [DOI] [PubMed] [Google Scholar]
  12. Ray A., Reynolds J. A., Polet H., Steinhardt J. Binding of large organic anions and neutral molecules by native bovine serum albumin. Biochemistry. 1966 Aug;5(8):2606–2616. doi: 10.1021/bi00872a019. [DOI] [PubMed] [Google Scholar]
  13. Razin A., Riggs A. D. DNA methylation and gene function. Science. 1980 Nov 7;210(4470):604–610. doi: 10.1126/science.6254144. [DOI] [PubMed] [Google Scholar]
  14. Sager R., Kovac P. Pre-adipocyte determination either by insulin or by 5-azacytidine. Proc Natl Acad Sci U S A. 1982 Jan;79(2):480–484. doi: 10.1073/pnas.79.2.480. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Santi D. V., Garrett C. E., Barr P. J. On the mechanism of inhibition of DNA-cytosine methyltransferases by cytosine analogs. Cell. 1983 May;33(1):9–10. doi: 10.1016/0092-8674(83)90327-6. [DOI] [PubMed] [Google Scholar]
  16. Starzyk R. M., Koontz S. W., Schimmel P. A covalent adduct between the uracil ring and the active site of an aminoacyl tRNA synthetase. Nature. 1982 Jul 8;298(5870):136–140. doi: 10.1038/298136a0. [DOI] [PubMed] [Google Scholar]
  17. Taylor S. M., Jones P. A. Changes in phenotypic expression in embryonic and adult cells treated with 5-azacytidine. J Cell Physiol. 1982 May;111(2):187–194. doi: 10.1002/jcp.1041110210. [DOI] [PubMed] [Google Scholar]
  18. Taylor S. M., Jones P. A. Mechanism of action of eukaryotic DNA methyltransferase. Use of 5-azacytosine-containing DNA. J Mol Biol. 1982 Dec 15;162(3):679–692. doi: 10.1016/0022-2836(82)90395-3. [DOI] [PubMed] [Google Scholar]
  19. Woodbury C. P., Jr, von Hippel P. H. On the determination of deoxyribonucleic acid-protein interaction parameters using the nitrocellulose filter-binding assay. Biochemistry. 1983 Sep 27;22(20):4730–4737. doi: 10.1021/bi00289a018. [DOI] [PubMed] [Google Scholar]
  20. Yoo O. J., Agarwal K. L. Isolation and characterization of two proteins possessing Hpa II methylase activity. J Biol Chem. 1980 Jul 10;255(13):6445–6449. [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES